The long term goal of this proposed investigation is to investigate the role of the nuclear modifier genes in the phenotypic expression of hearing impairment associated with mitochondrial DNA mutations and is to explore a therapeutic intervention for maternally inherited disorders. Mitochondrial 12S rRNA A1555G mutation has been found in many families of various ethnic origins with variable penetrance and expressivity of aminoglycoside-induced and non-syndromic hearing loss. Our previous studies indicated that the A1555G mutation is a primary factor for the development of deafness and nuclear modifier genes modulate the expression of deafness associated with this mutation. Recently, an interesting model for nuclear-mtDNA interaction for the phenotypic manifestation of A1555G mutation has been proposed. In the yeast, the mutant alleles of MTO1 or MSS1 or MTO2, involved in mitochondrial tRNA modification, manifest a respiratory-deficient phenotype only when coupled with the 15S rRNA PR454 mutation corresponding to the A1555G mutation. We showed that human GTPBP3 (homolog of MSS1), MTO1 and TRMT1 (homolog of MTO2) cDNA can restore the respiratory deficient phenotype of yeast mss1, mto1 or mto2 cells carrying the PR454 mutation, indicating that the functions of those proteins are evolutionarily conserved. These suggest that MSS1, MT01 or MTO2-like modifiers may influence the phenotypic expression of A1555G mutation. Especially, Trmt1, highly evolutionally conserved protein, is implicated to be involved in 2-thiouridine modification at position 34 of tRNALys, tRNAGlu and tRNAGln. Sequencing of TRMT1 in 392 members of an Arab-Israeli family and other Spanish/Italian families revealed a missense mutation (G28T) altering an invariant amino-acid residue (A10S) in the mitochondrial targeting sequence. In particular, subjects carrying both homozygous A10S and A1555G mutations exhibited deafness. This mutation does not affect importing of Trmt1 precursors. Strikingly, the homozygous A10S mutation causes defects in mitochondrial tRNA metabolisms, specifically reducing the steady-state levels of mitochondrial tRNAs. We hypothesize that TRMT1 is involved in the mitochondrial tRNA modification. As a result, these defects may contribute to the impairment of mitochondrial protein synthesis. Thus, the resultant biochemical defects aggravate the mitochondrial dysfunction associated with A1555G mutation, exceeding the threshold for expressing deafness phenotype. As a result, the mutated Trmtl, acting as a modifier factor, modulates the phenotypic manifestation of deafness-associated 12S rRNA A1555G mutation. These hypotheses will be addressed by the following specific aims. 1) Evaluate the effect of the A10S mutation in TRMT1 on mitochondrial dysfunction associated with the A1555G mutation. 2) Biochemical and expression characterization of mammalian mitochondrial Ttmt1 3) Assessment of the effects of the knocking-down TRMT1 on mitochondrial dysfunction associated with the A1555G mutation. Success of this project will define the role of nuclear genes in the pathogenesis of maternally inherited deafness. This, in turn, provides new insights into the molecular mechanism of maternally inherited deafness as well as other disease process. Also, this success will give rise to a deepen understanding the mechanism of the interaction between the nuclear and mitochondrial genome. In addition, the success of this research will contribute significantly to the development of animal models for therapeutic interventions.